Human epidemiology and mouse infection models indicate that estrogen is a potent modulator of immune cell function, responsible for many gender differences in immunity to respiratory infection. Little data exist identifying specific mechanisms by which estrogen improves lung defense. Alveolar macrophages (AMO), resident phagocytes of the terminal airways, are critical first line effectors of cellular immunity in the alveoli. We postulate that innate immune differences between the genders most likely act at this initial defense point. Preliminary data indicate that estrogen increases AMO bacterial killing through NOS3 activation and NO production. This proposal delineates experiments designed to (i) determine the mechanism(s) of AMO NOS3 activation in response to estrogen and (ii) examine NOS3-derived NO signaling in estrogen-treated AMO. The first goal will be achieved using fluorescent and biochemical assays of calcium and phosphorylation signaling pathways, including antibody-based, proteomic, and inhibitor and knockout approaches. The second goal will be accomplished using proteomic techniques to identify new protein S-nitrosylation targets in estrogen-treated immortalized wild-type and GSNOR-knockout AMO. [unreadable] [unreadable] [unreadable]